Monoclonal antibodies directed against the envelope glycoproteins of membrane viruses such as human immunodeficiency virus type 1 (HIV-1), influenza, and ebolavirus are essential reagents for deciphering mechanisms of viral entry, and identifying epitopes for immunotherapy or vaccine development. Recent work in antibody engineering has demonstrated that specific, high-affinity antibodies can be isolated from simple phage display libraries in which diversity at the antibody complementarity determining regions (CDRs) is encoded by designed, synthetically-derived oligonucleotides ('synthetic antibodies'). Therefore, the synthetic antibody approach circumvents many limitations of traditional antibody isolation methods thereby expanding the scope and specificity with which antigens may be targeted. The overall goal of this proposal is to develop enabling synthetic antibody technologies for applications in the study of viral membrane fusion. In Aim 1, we propose to develop synthetic antibody libraries focused toward viral antigens based on the promiscuous germline segment VH1-69. Many viral antibodies borne from this progenitor exhibit similar modes of interaction with their viral antigens, suggesting that VH1-69 could serve as a scaffold for development of virus- specific synthetic antibody libraries. We will produce VH1-69-based synthetic antibody libraries and screen them against viral targets to evaluate this hypothesis. In Aim 2, we propose to identify conformation-specific antibodies that target the membrane-proximal external region (MPER) of HIV-1 gp41. The MPER is the target of several broadly neutralizing antibodies (NAbs) and therefore the subject of intense investigation for vaccine development. We will identify synthetic antibodies against structurally-constrained MPER peptides, and then characterize neutralization potency of these antibodies to gain insight into the role of MPER epitope conformation on inhibition of membrane fusion. In Aim 3, we propose to isolate synthetic antibodies against the putative fusion intermediates of ebolavirus GP1 and GP2. In comparison to HIV-1 and influenza, relatively few antibodies against GP1 and GP2 have been isolated which has greatly impeded mechanistic understanding of membrane fusion for this virus. Our approach will overcome limitations of other antibody isolation methods that have failed to produce GP1- or GP2-specific antibodies, and yield novel reagents for dissection of fusion intermediates and potential immunotherapeutics.